Optical Activation of Photoswitchable TRPC Ligands in the Mammalian Olfactory System Using Laser Scanning Confocal Microscopy.

The transient receptor potential canonical (TRPC) ion channels play important biological roles, but their activation mechanisms are incompletely understood. Here, we describe recent methodological advances using small molecular probes designed for photopharmacology of TRPC channels by focusing on results obtained from the mouse olfactory system. These studies developed and used photoswitchable diacylglycerol (DAG) analogs for ultrarapid activation of native TRPC2 channels in vomeronasal sensory neurons and type B cells of the main olfactory epithelium. Further studies investigated the role of TRPC5 channels in prolactin regulation of dopamine neurons in the arcuate nucleus of the hypothalamus. Here, the first photoswitchable TRPC5 modulator, BTDAzo, was developed and shown to control endogenous TRPC5-based neuronal Ca2+ responses in mouse brain slices. Thus, photoswitchable reagents are rapidly gaining widespread recognition for investigating various types of TRPC channels including TRPC2, TRPC3, TRPC5, and TRPC6, enabling to gain new insights into the gating mechanisms and functions of these channels.

BTDAzo: A Photoswitchable TRPC5 Channel Activator.

Photoswitchable reagents can be powerful tools for high-precision biological control. TRPC5 is a Ca2+ -permeable cation channel with distinct tissue-specific roles, from synaptic function to hormone regulation. Reagents giving spatiotemporally-resolved control over TRPC5 activity may be key to understanding and harnessing its biology. Here we develop the first photoswitchable TRPC5-modulator, BTDAzo, to address this goal. BTDAzo can photocontrol TRPC5 currents in cell culture, as well as controlling endogenous TRPC5-based neuronal Ca2+ responses in mouse brain slices. BTDAzos are also the first reported azo-benzothiadiazines, an accessible and conveniently derivatised azoheteroarene with strong two-colour photoswitching. BTDAzo's ability to control TRPC5 across relevant channel biology settings makes it suitable for a range of dynamically reversible photoswitching studies in TRP channel biology, with the aim to decipher the various biological roles of this centrally important ion channel.

A diacylglycerol photoswitching protocol for studying TRPC channel functions in mammalian cells and tissue slices.

Small molecular probes designed for photopharmacology and opto-chemogenetics are rapidly gaining widespread recognition for investigations of transient receptor potential canonical (TRPC) channels. This protocol describes the use of three photoswitchable diacylglycerol analogs-PhoDAG-1, PhoDAG-3, and OptoDArG-for ultrarapid activation and deactivation of native TRPC2 channels in mouse vomeronasal sensory neurons and olfactory type B cells, as well as heterologously expressed human TRPC6 channels. Photoconversion can be achieved in mammalian tissue slices and enables all-optical stimulation and shutoff of TRPC channels. For complete details on the use and execution of this protocol, please refer to Leinders-Zufall et al. (2018).

Membrane potential manipulation with visible flash lamp illumination of targeted microbeads.

The electrical membrane potential (Vm) is a key dynamical variable of excitable membranes. Despite the tremendous success of optogenetic methods to modulate Vm with light, there are some shortcomings, such as the need of genetic manipulation and limited time resolution. Direct optical stimulation of gold nanoparticles targeted to cells is an attractive alternative because the absorbed energy heats the membrane and, thus, generates capacitive current sufficient to trigger action potentials [1, Carvalho-de-Souza et al., 2015]. However, focused laser light is required and precise location and binding of the nanoparticles cannot be assessed with a conventional microscope. We therefore examined a complementary method to manipulate Vm in a spatio-temporal fashion by non-focused visible flashlight stimulation (Xenon discharge lamp, 385-485 nm, ∼500 µs) of superparamagnetic microbeads. Flashlight stimulation of single beads targeted to cells resulted in transient inward currents under whole-cell patch-clamp control. The waveform of the current reflected the first time derivative of the local temperature induced by the absorbed light and subsequent heat dissipation. The maximal peak current as well as the temperature excursion scaled with the proximity to the plasma membrane. Transient illumination of light-absorbing beads, targeted to specific cellular sites via protein-protein interaction or direct micromanipulation, may provide means of rapid and spatially confined heating and electrical cell stimulation.

Non-photonic sensing of membrane-delimited reactive species with a Na+ channel protein containing selenocysteine.

Photonic experiments are of key importance in life sciences but light-induced side effects are serious confounding factors. Here we introduce roNaV2, an engineered voltage-gated Na+ channel harboring a selenocysteine in its inactivation motif, as a non-photonic, sensitive, gateable, and reversible sensor for membrane-delimited reactive species. roNaV2 allows for the assessment of chemical modification induced in fluorescence microscopy settings with high sensitivity and time resolution and it demonstrates the usefulness of ion channels as highly sensitive reporters of membrane processes.

Impact of higher-order heme degradation products on hepatic function and hemodynamics.

BACKGROUND & AIMS: Biliverdin and bilirubin were previously considered end products of heme catabolism; now, however, there is evidence for further degradation to diverse bioactive products. Z-BOX A and Z-BOX B arise upon oxidation with unknown implications for hepatocellular function and integrity. We studied the impact of Z-BOX A and B on hepatic functions and explored their alterations in health and cholestatic conditions. METHODS: Functional implications and mechanisms were investigated in rats, hepatocytic HepG2 and HepaRG cells, human immortalized hepatocytes, and isolated perfused livers. Z-BOX A and B were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in acute and acute-on-chronic liver failure and hereditary unconjugated hyperbilirubinemia. RESULTS: Z-BOX A and B are found in similar amounts in humans and rodents under physiological conditions. Serum concentrations increased ∼20-fold during cholestatic liver failure in humans (p<0.001) and in hereditary deficiency of bilirubin glucuronidation in rats (p<0.001). Pharmacokinetic studies revealed shorter serum half-life of Z-BOX A compared to its regio-isomer Z-BOX B (p=0.035). While both compounds were taken up by hepatocytes, Z-BOX A was enriched ∼100-fold and excreted in bile. Despite their reported vasoconstrictive properties in the brain vasculature, BOXes did not affect portal hemodynamics. Both Z-BOX A and B showed dose-dependent cytotoxicity, affected the glutathione redox state, and differentially modulated activity of Rev-erbα and Rev-erbß. Moreover, BOXes-triggered remodeling of the hepatocellular cytoskeleton. CONCLUSIONS: Our data provide evidence that higher-order heme degradation products, namely Z-BOX A and B, impair hepatocellular integrity and might mediate intra- and extrahepatic cytotoxic effects previously attributed to hyperbilirubinemia. LAY SUMMARY: Degradation of the blood pigment heme yields the bile pigment bilirubin and the oxidation products Z-BOX A and Z-BOX B. Serum concentrations of these bioactive molecules increase in jaundice and can impair liver function and integrity. Amounts of Z-BOX A and Z-BOX B that are observed during liver failure in humans have profound effects on hepatic function when added to cultured liver cells or infused into healthy rats.

Sodium channels as gateable non-photonic sensors for membrane-delimited reactive species.

Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNaV). roNaV can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNaV has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNaV is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed have revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000 ms.